Abstract / Project Summary Hepatitis B virus (HBV) infects ~240 million people chronically and can progress to cirrhosis and hepatocellular carcinoma (HCC), the third leading cause of cancer mortality. Current drug therapies to treat HBV cannot eliminate the persistent covalently closed circular (ccc)DNA, which thus requires lifetime drug therapy. Therefore, the discovery of novel HBV drugs that can eliminate cccDNA is an active area of pharmaceutical drug development. However, such efforts are hampered by the lack of physiologically-relevant model systems that recapitulate critical features of the disease pathogenesis, such as interactions between hepatocytes and relevant liver non-parenchymal cell types (NPCs), and the ability to evaluate patient-specific outcomes. Conducting tests on the chimpanzee, the only animal model for HBV, is prohibitively expensive, severely restricted in the US and Europe, and does not fully mimic human HBV pathogenesis. Thus, human liver culture platforms are the most widely used model for HBV studies. While primary human hepatocytes (PHHs) are the gold standard for HBV studies, they are a scarce resource that does not suffice for high-throughput drug screening and to elucidate the genetic basis of HBV pathogenesis. On the other hand, induced pluripotent stem cell-derived human hepatocyte- like cells (iHeps) are an important patient-specific source from an expandable precursor, thus mitigating many of the limitations with PHHs. We and others have shown that 2D cultures of iHeps can be infected with HBV and used to test drugs. However, absorbed extracellular matrix (ECM) proteins in 2D cultures do not allow adequate elucidation of ECM reorganization/re-modeling during HBV-induced fibrosis progression. Furthermore, existing platforms lack the relevant liver NPCs that interact with HBV-infected hepatocytes in vivo to modulate infection, inflammation, and fibrosis. We have recently pioneered microscale 3D collagen microgels containing iHeps that display adult-like liver functions, including drug metabolism enzyme activities, for several weeks in vitro when co-cultured with primary liver sinusoidal endothelial cells (LSECs), herein referred to as ?microtissues?. In this proposal, we will test our novel hypothesis that iHep/LSEC 3D microtissues, which display adult-like liver functions, can a) be infected chronically (weeks) with HBV with higher infection levels, spread, and amplification than existing 2D/3D culture platforms (aim 1), and b) when augmented with hepatic stellate cells (HSCs) and Kupffer cells (KCs), display inflammatory and fibrotic signatures correlative of clinical outcomes (aim 2). Our proposal will yield a first-of-its-kind scalable 3D human liver platform containing iHeps and liver NPCs that displays chronic HBV infection while retaining the ability to adequately metabolize drugs for screening. In the future, our HBV platform can be used to study mechanisms underlying hepatocyte-NPC interactions towards discovering novel druggable targets, the inclusion of patient-matched adaptive immune cells to model interaction with resident liver cell types, and gene editing to elucidate genetic determinants of HBV progression.